Therapeutic effect of nerve growth factor supplying implant on the inferior alveolar nerve regeneration and osseointegration

Abstract

목적: 임플란트 시술이 대중화되면서 하치조신경 손상에 의한 감각 이상도 급격히 증가하고 있으며 그 중요성도 점점 부각되고 있다. 본 연구에서는 하치조신경 손상 수복 부위에 신경성장인자(NGF)를 공급할 수 있도록 특별히 제작된 치과 임플란트를 이용하여 NGF를 공급했을 때 신경 재생에 대한 효과를 평가하였으며, NGF 공급과 개조된 임플란트의 디자인이 임플란트의 골유착에 미치는 영향을 검증하고자 하였다.

재료 및 방법: 18주령 성견 (beagle dog, n=9)을 전신 마취시킨 후 양측 하치조신경을 노출시키고 하악 제3소구치를 발거한 후 NGF 공급 임플란트를 즉시 식립하였다. 임플란트에 근접한 부위의 하치조신경을 절단한 후 단단 문합하고 mini osmotic pump 카테터를 임플란트에 연결하였다. 우측 하치조신경에는 2mL의 NGF 용액 (150μg/mL)이 그리고 좌측 하치조신경에는 동량의 PBS 용액이 담긴 pump를 연결하였다. 신경재생 평가를 위해 술전 및 술후 3, 6주에 needle electrode를 이용해 진폭과 잠복시간을 측정하였고 신경절단 전후 및 술후 6주에 hook electrode를 이용해 신경전도속도와 최대전압을 측정하였다. 술후 6주에 손상부를 중심으로 신경표본을 채취한 후 시편 중앙부를 횡분절 (cross section)하여 epon 포매 절편 toluidine blue로 염색 후 전체 축삭 수, 축삭밀도를 측정하고 대표 부위를 TEM표본 제작하여 축삭 수초의 두께 및 G-ratio를 측정하였다. 임상적으로는 식립된 임플란트의 염증 등 부작용을 관찰하였으며, 술후 6주째 임플란트 안정도 (ISQ)를 측정하였다. 그리고 6주 후 희생 시 임플란트를 주변 조직과 같이 채취하여 탈회, 비탈회 표본을 제작하고 이를 통해 bone-implant contact (BIC) 비율과 bone area (BA) 비율을 측정하였다.

결과: 전기신경생리측정에서 진폭과 잠복시간은 두 군 사이에 유의한 차이가 없었다. 신경전도속도에서는 6주째 NGF군의 중앙값(2.675 m/s)이 PBS군(1.892 m/s)보다 유의하게 높았으며 (p < 0.01), 최대전압도 같은 결과를 보였다 (NGF vs. PBS : 1.940 μV vs. 1.300 μV, p < 0.01). 마찬가지로 NGF군의 전체 축삭 수 및 축삭밀도 (4576.107 ± 270.413, 10707.458 ± 638.835/mm2)가 모두 PBS군 (3606.972 ± 242.876, 7899.781 ± 1063.625/mm2)에 비해 유의하게 높았지만 (p < 0.001), G-ratio는 두 군간 차이가 없었다. BIC 는 NGF군이 46.609±6.521 %, PBS군이 42.884±6.489 % 였으며, 두 군 간에 유의한 차이는 없었다. BA (NGF vs. PBS : 36.993±7.0434% vs. 33.327±6.551 %)와 6주 후 ISQ (NGF vs. PBS : 42.375±3.017 vs. 38.714±2.533)에서도 두 군간 유의한 차이가 관찰되지 않았다. NGF 공급 임플란트의 전체 생존률은 83.33% 이었으며 NGF 군은 88.89%, PBS군은 77.78%였다.

결론: 본 연구에서 NGF 공급 임플란트를 이용한 NGF 공급은 하치조신경의 재생을 도모하였으며 이러한 처치가 골유착을 저해하지 않았음을 알 수 있었다. 이러한 치료 방식은 신경재생과 골유착을 동시에 도모할 수 있기 때문에 임플란트 식립과 관련된 하치조신경 손상치료의 새로운 가능성을 제시할 수 있다고 사료되었다.

Purpose: Among the various complications of dental implant procedure, sensory disturbance caused by the damage of inferior alveolar nerve (IAN) is one of the most difficult and challenging issues with continually increasing incidence. The purpose of this study was to evaluate the functional regeneration of IAN by supplying nerve growth factor (NGF) using specially designed dental implant and its effect on the osseointegration of NGF-supplying implant.

Materials and Methods: Under general anesthesia with intravenous injection of the mixture of tiletamine and zolazepam, bilateral IAN of beagle dogs (n=9, 18 week-old, 10 ~ 12kg) was exposed, transected and directly repaired. NGF-supplying implants were installed just above the nerve damage site after the third premolar extraction and connected with osmotic pump by microcatheter. For experimental group (right IAN), total 300μg of NGF mixed with 2mL PBS solution was loaded in the pump and same amount of PBS for control group (left IAN). The amplitude and latency were measured with needle-type electrodes before surgery and at postoperative 3, 6 week. The conduction velocity (CV) and peak voltage (PV) were measured with custom-made hook type electrodes before transection, after nerve repair and at postoperative 6 week. At 6 week, nerve specimens were harvested and embedded to calculate axon count and density. After that, TEM analysis was done to measure the myelin thickness and G-ratio. Dental implants were observed clinically with checking the implant stability quotient (ISQ). After 6 weeks, each dental implant was prepared for the decalcified and non-decalcified sections for the measurement of bone-implant contact ratio (BIC) and the new bone area inside the thread area (BA).

Results: In the amplitude and latency, there were no significant differences between two groups. The median CV of NGF group (2.675 m/s) was significantly higher than that of PBS group (1.892 m/s) at 6 week (p < 0.01) and same tendency was also observed in PV (1.940 μV in NGF group and 1.300 μV in PBS group). The axon count of NGF group (4576.107±270.413) was higher than that of PBS group (3606.972±242.876) with significance (p < 0.001) and same as axon density (NGF group : 10707.458±638.835/mm2, PBS group : 7899.781±1063.625/mm2,p < 0.001). In myelin thickness, mean of NGF group (1.670±0.555 μm) was higher than that of PBS groups (1.173±0.388 μm) with significance (p < 0.01), but no significant difference was found in G-ratio between two groups (NGF vs. PBS, 0.594±0.110 vs. 0.635±0.092). The BIC of NGF group (46.609±6.521 %) was higher than that of PBS group (42.884±6.489 %), but no significant difference was found. The same tendency was found in the BA (36.993±7.0434 % in NGF group and 33.327±6.551 % in PBS group) and ISQ (42.375±3.017 in NGF group and 38.714±2.533 in PBS group). Overall survival rate of NGF-implant was 88.89% in NGF group (88.89%), and 77.78% in PBS group.

Conclusions: NGF group showed higher conduction velocity and peak voltage across the nerve repair with significance. Same tendencies were also observed in amplitude and latency of CNAP, but there were no significant differences between two groups. In histomorphometric analysis, NGF group exhibited significantly higher axon count, axon density and myelin thickness than those of PBS group, but there were no significant differecence in G-ratio, BIC and BA. Except three exfoliated implants, no significant difference was found in ISQ between NGF group and PBS group at 6 week. These results showed that NGF supplying via specially designed dental implant could promote the functional regeneration of IAN transection-repair injury. Furthermore, supplied NGF and the design of implant did not interrupt the osseointetegration of implant. This approach could be a novel technique in the treatment of IAN injury, in which nerve regeneration and prosthetic rehabilitation could be achieved simultaneously.